Site News  |   Monitor Keywords  |   Monitor Archive  |   Organizer  |   Account Info  |

Ligand activated transcriptional regulator proteins

Ligand activated transcriptional regulator proteins description/claims

This application is a divisional of allowed U.S. application Ser. No. 10/422,934, filed Apr. 23, 2003, which is a continuation of U.S. application Ser. No. 09/586,625, filed Jun. 2, 2000, now U.S. Pat. No. 7,329,728, which is a continuation-in-part of U.S. application Ser. No. 09/433,042, filed Oct. 25, 1999, now abandoned. The subject matter of each of U.S. application Ser. Nos. 09/433,042, 09/586,625 and 10/422,934 is herein incorporated by reference in its entirety.

Work described herein was supported by National Institutes of Health NIH Contract No. GM53910. The United States Government has certain rights in such subject matter.

An electronic version on compact disc (CD-R) of the Sequence Listing is filed herewith in duplicate (labeled Copy #1 and Copy #2), the contents of which are incorporated by reference in their entirety. The computer-readable file on each of the aforementioned compact discs, created on Jul. 18, 2008, is identical, 234 kilobytes in size, and titled 1227DSEQ.001.txt.

The field of this invention is the regulation of gene expression. In particular, ligand-activated fusion proteins (also referred to herein as chimeric regulators) and the use thereof for regulation of gene expression are provided. The fusion polypeptides contain a DNA binding domain containing one or a plurality of zinc finger polypeptide domains and a ligand binding domain (LBD) derived from an intracellular receptor.

Intracellular receptors are a superfamily of related proteins that mediate the nuclear effects of a variety of hormones and effector molecules, including steroid hormones, thyroid hormones and vitamins A and D. Members of this family of intracellular receptors are prototypical ligand activated transcription factors. These receptors contain two primary functional domains: a DNA binding domain (DBD) that contains about sixty-six amino acids and a ligand-binding domain (LBD) located in the carboxyl-terminal half of the receptor that has about 300 amino acids. The receptors are inactive in the absence of hormone (ligand) by virtue of association with inactivating factors, such as heat shock proteins. Upon ligand binding, the receptors dissociate from the inactivating complex and dimerize, which renders them able to bind to DNA and modulate transcription.

For example, for the steroid receptors, binding of a steroid hormone to its receptor results in receptor protein homodimerization and subsequent binding to the “steroid response element” (SRE) DNA sequence in nuclear DNA. Conformational changes in the receptor associated with ligand binding results in the recruitment of other transcriptional regulatory proteins, called co-activators, that regulate the transcription from promoters adjacent to the SRE binding sites.

Modified steroid hormone receptors have been developed for use for regulated expression of transgenes (see, e.g., U.S. Pat. No. 5,874,534 and published International PCT application No. WO 98/18925, which is based on U.S. provisional application Ser. No. 60/029,964) by modifying the ligand specificity of the LBD. In addition, the DNA binding domain of the receptor has been replaced with a non-mammalian DNA binding domain selected from yeast GAL4 DBD, a viral DBD and an insect DBD to provide for regulated expression of a co-administered gene containing a region recognized by the non-mammalian DBD. These constructs, however, have several drawbacks. The non-mammalian DBD is potentially immunogenic and the array of sequences recognized by these DBD is limited, thereby severely restricting gene targets.

Therefore, there remains a need for more versatile gene regulators. It is an object herein to provide polypeptides that function as versatile regulators of gene expression.

Polypeptides that function as ligand activated transcriptional regulators and nucleic acid molecules encoding such polypeptides are provided. The polypeptides are fusion proteins that are ligand activated transcriptional regulators that can be targeted to any desired endogenous or exogenous gene. Variants of the fusion protein can be designed to have different selectivity and sensitivity for endogenous and exogenous ligands.

Nucleic acid molecules encoding the fusion proteins, expression vectors containing the nucleic acids and cells containing the expression vectors are provided. The fusion protein or nucleic acids, particularly vectors, that encode the fusion protein can be introduced into a cell and, when expressed in the cell, regulate gene expression in a ligand-dependent manner.

Fusion Proteins

The fusion proteins provided herein contain a ligand binding domain (designated herein LBD) from an intracellular receptor, preferably a LBD that has modified ligand specificity compared to the native intracellular receptor from which the LBD originates, and a nucleic acid binding domain (designated herein DBD) that can be tailored for any desired specificity. The fusion proteins may also include a transcriptional regulating domain (designated herein TRD), particularly a repressor or activator domain. The domains are operatively linked whereby the resulting fusion protein functions as a ligand-regulated targeted transcription factor.

When delivered to the nucleus of a cell, the domains, which are operatively linked, together act to modulate the expression of a targeted gene, which may be a native gene in a cell or a gene that also is delivered to a cell. Hence the targeted gene can be an endogenous cellular gene or an exogenously supplied recombinant polynucleotide construct. The fusion protein may also include a transcriptional regulating domain that is selected to activate, enhance or suppress transcription of a targeted gene.

In one embodiment, the fusion protein is constructed from components highly similar to human proteins, preferably components that are about 80% more preferably about 85%, most preferably at least about 90% identical in amino acid sequence to the corresponding human domain. In another embodiment, the fusion protein binds to a naturally occurring gene and modulates the transcription of the naturally occurring gene in a ligand-dependent way. In another embodiment, the fusion protein binds to an exogenously supplied recombinant construct and modulates the transcription of the exogenously supplied recombinant construct in a ligand-dependent way.

• Provisional Patent
• Utility Patent

• What Is a Patent?
• What Is a Trademark or Servicemark?
• What Is a Copyright?
• Patent Laws